Project Summary Specific Aim 1. To identify key differentiation genes. We hypothesized that epigenetic dysregulation of neural crest stem cells and/or sympathoadrenal progenitors contributes to neuroblastoma initiation, tumorigenesis and progression. Moreover, by targeting critical chromatin regulators that keep neuroblastoma in a self-renewal state we should be able to suppress growth and induce differentiation. To do this we performed an epigenetic focused siRNA screen to identify genes that control NB cell proliferation and differentiation using a high-throughput, high content imaging screen. We identified 53 candidate genes whose loss of expression results in a decrease in the number of NB cells and of these, 16 also induce morphologic and biochemical evidence of differentiation. A secondary screen using additional siRNAs excluded genes which may have resulted from off-target effects of siRNAs. Four of the candidates had already been shown to affect NB cell growth and differentiation. To prioritize those hits that would be amenable to drug development, we performed an additional screen of a tool compound library of 20 small molecule inhibitors of chromatin regulators. We evaluated the growth and differentiation in 8 NB cell lines and 2 immortal, but not transformed cell lines after exposure for 7days to 8 different drug concentrations. The secondary chemical screen identified EZH2 and SETD8 as druggable NB targets. Specific Aim 2. To characterize molecular mechanisms of action of growth and differentiation genes Our group was the first to identify that EZH2 expression was elevated in NB tumors and functioned to suppress tumor suppressor genes like CASZ1 as well as differentiation genes (Wang et al Cancer Res. 2012). We have continued our study of EZH2 as it was also found in our high-content imaging epigenetic screen that loss of EZH2 expression led to a decrease in cell growth and an increase in differentiation of NB cells. We collaborated with Dr. Kim Stegmaier's group at Dana-Farber Cancer Center and Boston Children's Hospital to examine more broadly the role of EZH2 in NB. In this study which was recently published we found using a CRISPR library that NB cell growth was dependent on the PRC2 complex and loss of any component of the PRC2 complex of which EZH2 contains the methylase activity resulted in loss of NB cell growth. Additionally clinically relevant small molecule inhibitors of EZH2 showed activity in NB cell line and NB Patient Derived xenografts as single agents and synergistic activity with HDAC inhibitors such as Panobinostat. Detailed mechanistic studies revealed that a driver of EZH2 expression was MYCN which is amplified in some 30% of high-risk NB patients and EZH2 functioned to suppress differentiation associated gene programs. A pediatric phase I evaluation of Tazemetostat is ongoing for INI deleted rhabdoid tumors and synovial sarcomas, and our study provides convincing pre-clinical rationale for the assessment in NB patients. This study was recently published in Chen, L. et al J. Clin. Invest. 128:458-462, 2018. Current studies have been aimed at defining the higher order chromatin structure when Neuroblastoma cells switch from a self-renewal state to implement a differentiation program. Using our Retinoic Acid induced differentiation model we have identified 4 clusters of Super-enhancer regulated genes that are dynamically regulated during the differentiation process. Current studies are analyzing functional requirements of different genes in the differentiation process.